Extensive tyrosine nitration in human myocardial inflammation: Evidence for the presence of peroxynitrite

Abstract

OBJECTIVES: Production of nitric oxide via the cytokine-mediated activation of myocardial inducible nitric oxide synthase decreases myocardial contractility. Whether myocardial dysfunction is mediated directly by nitric oxide or indirectly through the formation of secondary reaction products, such as peroxynitrite, has not been established. Peroxynitrite, but not nitric oxide, reacts with the phenolic ring of tyrosine to form the stable product 3-nitro-L-tyrosine. Demonstration of tissue nitrotyrosine residues, therefore, infers the presence of peroxynitrite or related nitrogen-centered oxidants. DESIGN: Retrospective analysis of human autopsy specimens. SETTING: University pathology and basic science laboratories. PATIENTS: Formalin-fixed, paraffin-embedded myocardial tissue samples were obtained from 11 patients with a diagnosis of sepsis, seven patients with a diagnosis of viral myocarditis, and five control patients without clinical or pathologic cardiac disease. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Specific antibodies to nitrotyrosine were utilized to detect nitrotyrosine residues in human autopsy specimens. Cardiac tissue obtained from patients with myocarditis or sepsis demonstrated intense nitrotyrosine immunoreactivity in the endocardium, myocardium, and coronary vascular endothelium and smooth muscle. In contrast, connective tissue elements were without appreciable immunohistochemical staining. Nitrotyrosine antibody binding was blocked by coincubation with nitrotyrosine or nitrated bovine serum albumin, but not by aminotyrosine, phosphotyrosine, or bovine serum albumin. In situ reduction of tissue nitrotyrosine to aminotyrosine by sodium hydrosulfite also blocked antibody binding. Densitometric analysis of nitrotyrosine immunoreactivity demonstrated significantly higher values for specimens from myocarditis and sepsis patients when compared with control tissue specimens. CONCLUSION: These results demonstrate the formation of peroxynitrite within the myocardium during inflammatory disease states, suggesting a role for peroxynitrite in inflammation-associated myocardial dysfunction.